Project 1: Regulation of microtubule growth downstream of Rac1 GTPase by CLIP-170[unreadable] Personnell: Yukako Nishimura[unreadable] [unreadable] Microtubule dynamics are regulated by the Rho small GTPase[unreadable] family during cell migration. The active form of Rac1 induces[unreadable] lamellipodial protrusion and microtubule growth at the leading[unreadable] edge, however, it is unclear how Rac1 regulates microtubule[unreadable] dynamics and coordinates this with cell migration. CLIP170 is[unreadable] a member of the +TIP family of microtubule-associated proteins,[unreadable] which bind to tips of growing microtubules. Disruption of[unreadable] CLIP 170 in cells resulted in reduced microtubule rescue[unreadable] during rapid shortening, suggesting that CLIP170 acts to promote[unreadable] microtubule rescue. Since promotion of microtubule[unreadable] rescue may result in promotion of microtubule net growth, we[unreadable] sought to test the hypothesis that Rac1 mediated promotion of[unreadable] microtubule growth requires CLIP170. We depleted CLIP170[unreadable] protein in human cultured cells (U2OS) using siRNA and examined[unreadable] microtubule dynamics by monitoring either fluorescent-[unreadable] labelled tubulin or EB1-GFP. Interestingly, depletion of[unreadable] CLIP170 suppressed the microtubule growth induced by constitutively[unreadable] active Rac1. This result suggests that Rac1 promotes[unreadable] microtubule growth through regulation of CLIP170 activity[unreadable] at the leading edge of migrating cells.[unreadable] [unreadable] This work was presented as poster presentations in 2009 by Yukako at the Gordon Research Conference on Motile and Contractile Systems, and at the American Society for Cell Biology Annual Meeting.[unreadable] [unreadable] [unreadable] [unreadable] Project 2: Regulation of Microtubule dynamics in migrating cells by Kinesin microtubule depolymerases.[unreadable] Personell: Kenneth Myers[unreadable] Maintenance of cell polarity during directed migration of metastatic cells depends upon the dynamic nature of the microtubule cytoskeleton. In migrating cells, an asymmetry in MICROTUBULE dynamics is perpetuated by Rho-GTPase-mediated MICROTUBULE leading-edge assembly, and poorly characterized cell-rear disassembly signals. Depolymerizing kinesins (Kin-Is), including MCAK and Kif18a, are unique kinesins that work to enhance MICROTUBULE disassembly and thus are possible candidates to regulate cell polarity during cell migration. I will test the hypothesis that Rho-GTPase signaling to the Kin-Is MCAK and/or Kif18a promotes MICROTUBULE disassembly in the rear of migrating cells, and that this is necessary for cell polarization and directed migration. List aims here Experiments will identify the roles of Kin I-mediated control of MICROTUBULE dynamics in a migrating cell model. shRNA depletion and expression of phospho-mutant Kin I constructs will test Kin I regulation, MICROTUBULE stability, and migratory aberrations. Finally, the effects of Rho-GTPase signaling on Kin I activity in the rear and leading edge will be determined to characterize how Kin Is control region-specific MICROTUBULE dynamics. It is anticipated that manipulating Kin I activity and thereby modulating MICROTUBULE dynamics, will directly influence polarized migration.